Abstract This proposal focuses on the role of the heat shock proteins (HSPs) in the initiation of immune responses. Evidence gathered over three decades shows that six HSPs namely gp96, hsp70, hsp90, calreticulin, hsp110 and grp170, in the extracellular environment, prime T cell responses specific to antigens they chaperone. Many aspects regarding the mechanism of how these HSPs prime immune responses remain unknown, even though HSPs are now being explored in clinical immunotherapy of patients with cancer and infectious disease. A major advance in this regard was our identification of CD91 as a cell surface receptor for HSPs. CD91 serves as an endocytic and signaling receptor for gp96, hsp90, hsp70 and calreticulin. Recently, we and others have observed differences in the immune outcomes elicited by gp96, hsp70 and calreticulin despite the fact that they all interact with and are dependent on the receptor CD91. Signals transduced by CD91 downstream appear to be different when each HSP engages the receptor, leading to differential patterns of co-stimulation. This includes differential patterns of cytokines and expression of co-stimulatory molecules elicited by the antigen presenting cell (APC). Therein, HSPs can prime Th1, Th2, Treg or Th17 responses. We thus formulate the following overall hypothesis; ?HSPs in the extracellular environment each interact with CD91 on APCs in a unique way to activate distinct pathways including the inflammasome for co-stimulation?. The first aim of the proposal examines the interaction of gp96/hsp90, hsp70, or calreticulin with various ligand binding domains of CD91 expressed on APCs. The interaction of HSPs with CD91 is necessary for both the internalization of the HSP-peptide complex and for transduction of signals within the APC. Since there is little apparent structural homology between the HSPs, and a lack of common interacting modules, the structural interaction of each HSP with CD91 must be unique, initiating signaling cascades and individual cytokine patterns. We shall examine the binding domains on CD91 for each of the respective HSPs. In an extension of this aim, we shall identify adaptor proteins that associate with the phosphorylated cytoplasmic domain of CD91 with respect to each HSP ligand, providing an insight into the different signaling pathways that are initiated. In the second aim, we focus on cellular outcomes of the HSP-CD91 interaction, specifically cytokine release and related adjuvanticity. IL-1?? a cytokine that is commonly released from HSP-stimulated APCs is explored. We propose that HSPs are capable of activating components of the inflammasome, known to be necessary for IL-1? release. Results from this proposal are expected to inform heavily on the next generation of HSP-based vaccines for cancer and infectious disease being developed.